A scanning electron microscope (SEM) is an apparatus that has been widely used not only for morphological observation on an inorganic material sample or an organic material sample but also for observation on a biological sample, and is suitable for observing microorganisms, such as bacteria and viruses, which cannot be observed by the unaided eye.
Unfortunately, there are problems in that such a biological sample is susceptible to damage due to electron beam irradiation and it is difficult to acquire a high contrast image. For SEM observation on a biological sample, typically, a sample as an observation target is pretreated; in the pretreatment, the sample is fixed with formaldehyde or the like, and coated on the surface with gold, platinum, carbon or the like or stained with heavy metal or the like. Measures are taken where the treatment reduces damage to the sample due to an electron beam and increases the contrast.
Recently, a method has been developed that acquires a high contrast image without the aforementioned coating or staining on a biological sample (see Patent Literature 1 and Non Patent Literature 1). In this method, an observation target sample is caused to adhere to the undersurface (rear surface) of a thin sample supporting film (carbon film), and irradiated with an electron beam accelerated at a relatively low voltage from the top surface (front surface) of the sample supporting film. The electron beam incident on the sample supporting film spreads in the supporting film while diffusing. Electrons, reaching the undersurface of the supporting film and therearound, cause secondary electrons to be emitted. The secondary electrons are absorbed by the observation target sample adhering to the undersurface of the supporting film, thereby forming contrast.
The energy of such secondary electrons is significantly low, which is about 10 eV. The electrons cause almost no damage even if being absorbed by a biological sample. Furthermore, the degree of absorption of the electron beam is represented as-is as contrast. Accordingly, a significantly clear and high contrast SEM image can be acquired. Such an observation condition is referred to as an “indirect secondary electron contrast condition”.
An X-ray microscope has also been proposed having a configuration that causes an electron beam to enter a metal thin film, irradiates an observation target sample with X-rays emitted from the metal thin film, and acquires an X-ray image (Patent Literatures 2 and 3). Such an X-ray microscope emits X-rays which has a high penetrating power of radiation and with which an observation target sample is to be irradiated. Accordingly, there is an advantageous effect of allowing the inner structure of a sample to be observed.